Combined use of immunocytochemistry and in situ hybridization to study beta thyroid-stimulating hormone gene expression in pituitaries of hypothyroid rats.
Immunocytochemistry and in situ hybridization were used to demonstrate beta thyroid-stimulating hormone (TSH) immunoreactivity and beta TSH messenger RNA (mRNA) in the same tissue section of rat pituitary. Sections (10 micronsP of 4% paraformaldehyde-fixed pituitaries from surgically thyroidectomized and intact male rats were first hybridized with a 32P-labelled complementary RNA beta TSH probe and were then immunostained for beta TSH. In both sets of animals beta TSH mRNA and beta TSH immunoreactivity were simultaneously localized to many thyrotrophs, although there was considerable heterogeneity in mRNA labelling intensity between individual cells. In hypothyroid rats more cells were positive with both probe and antiserum, and the intensity of mRNA labelling in most quantified by direct apposition of hybridized sections to autoradiography film, and the image grey levels were measured using an image analyser to convert them into equivalent amounts of radioactivity. In hypothyroid rats this amount was 22 times greater than in controls (control 4.77 +/- 0.84 attomoles mm-2; hypothyroid 107.06 +/- 14.2 attomoles mm-2; mean +/- SEM p less than 0.001). In situ hybridization has been used in combination with immunocytochemistry, to demonstrate beta TSH and mRNA and immunoreactivity at a cellular level in rats with different thyroid status.
Steel
J H
JH
Department of Histochemistry, Royal Postgraduate Medical School, London.
O'Halloran
D J
DJ
Jones
P M
PM
Van Noorden
S
S
Chin
W W
WW
Bloom
S R
SR
Polak
J M
JM
D000818:Animals; D015342:DNA Probes; D005786:Gene Expression Regulation; D007037:Hypothyroidism / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D008297:Male; D009693:Nucleic Acid Hybridization; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D013972:Thyrotropin / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics
D015342:DNA Probes; D012333:RNA, Messenger; D013972:Thyrotropin